icell Cardiac Progenitor Cells Prototype User s Guide

Transcription

1 icell Cardiac Progenitor Cells Prototype User s Guide icell Cardiac Progenitor Cells User s Guide 1

2 Trademarks icell and MyCell are registered trademarks, and Cellular Dynamics and the logo are trademarks of Cellular Dynamics International, Inc. and may not be used without the express written permission of Cellular Dynamics International, Inc. (CDI). All other brands, product names, company names, trademarks, and service marks are the properties of their respective owners. Restrictions and Liabilities This document is provided as is. CDI assumes no responsibility for any typographical, technical, or other inaccuracies in this document. CDI reserves the right to periodically change information that is contained in this document; however, CDI makes no commitment to provide any such changes, updates, enhancements, or other additions to this document to you in a timely manner or at all. THE icell CARDIAC PROGENITOR CELLS ARE PROVIDED AS IS TO YOU. TO THE FULLEST EXTENT PERMITTED BY APPLICABLE LAW, CDI MAKES NO, AND DISCLAIMS ALL, REPRESENTATIONS, WARRANTIES, CONDITIONS OR COVENANTS, EXPRESS OR IMPLIED, WITH RESPECT TO ANY PRODUCT REFERENCED HEREIN OR PERFORMANCE OF ANY SERVICES REFERENCED HEREIN, INCLUDING BUT NOT LIMITED TO, ANY EXPRESS OR IMPLIED WARRANTIES OR CONDITIONS OF FITNESS FOR A PARTICULAR PURPOSE, NON-INFRINGEMENT, MERCHANTABILITY, DURABILITY, TITLE, OR RELATED TO THE PERFORMANCE OR NON-PERFORMANCE OF ANY PRODUCT REFERENCED HEREIN OR PERFORMANCE OF ANY SERVICES REFERENCED HEREIN). This document might contain references to third-party sources of information, hardware or software, products or services and/or third-party websites (collectively the Third-Party Information ). CDI does not control, and is not responsible for, any Third-Party Information, including, without limitation the content, accuracy, copyright compliance, compatibility, performance, trustworthiness, legality, decency, links, or any other aspect of Third-Party Information. The inclusion of Third-Party Information in this document does not imply endorsement by CDI of the Third-Party Information or the third party in any way. CDI does not in any way guarantee or represent that you will obtain satisfactory results from using icell Cardiac Progenitor Cells as described herein. You assume all risk in connection with your use of icell Cardiac Progenitor Cells. Conditions of Use icell Cardiac Progenitor Cells are for life science research use only and subject to the use restrictions contained in Appendix A. You are responsible for understanding and performing the protocols described within this guide. CDI does not guarantee any results you may achieve. These protocols are provided as CDI s recommendations based on its use and experience with icell Cardiac Progenitor Cells. Origin icell Cardiac Progenitor Cells are manufactured in the United States of America. Copyright Notice 2017 Cellular Dynamics International, Inc. All rights reserved. This document may not be reproduced, distributed, modified or publicly displayed without the express written permission of Cellular Dynamics International, Inc. Revision History Version 0.3: April 2017 Version 0.2: September 2015 Version 0.1: December 2014

3 Table of Contents Before You Begin... ii Chapter 1. Introduction... 1 Components Supplied by Cellular Dynamics... 2 Required Equipment and Consumables... 2 Technical Support and Training... 3 Workflow Diagram... 4 Chapter 2. Handling and Storage... 5 Chapter 3. Preparing Cell Culture Surfaces... 6 Chapter 4. Preparing the Medium... 7 Chapter 5. Thawing icell Cardiac Progenitor Cells... 8 Chapter 6. Plating icell Cardiac Progenitor Cells for Cardiac Proliferation Chapter 7. Plating icell Cardiac Progenitor Cells for Cardiomyocyte Differentiation Appendices Appendix A. Intellectual Property Rights, Use Restrictions, and Limited License Appendix B. Product Provided AS IS Appendix C. Limited Liability icell Cardiac Progenitor Cells Prototype User s Guide i

4 Before You Begin Notes Immediately transfer the frozen vials to liquid nitrogen storage. Read this entire icell Cardiac Progenitor Cells Prototype User s Guide before handling or using icell Cardiac Progenitor Cells. icell Cardiac Progenitor Cells are for life science research use only. See Appendix A for more information and other restrictions. A Safety Data Sheet (SDS) for dimethyl sulfoxide (DMSO), in which icell Cardiac Progenitor Cells are frozen, is available online at or on request from Cellular Dynamics International. Only technically qualified individuals experienced in handling DMSO and human biological materials should access, use, or handle icell Cardiac Progenitor Cells. ii Cellular Dynamics International, Inc.

5 Notes Chapter 1. Introduction Cellular Dynamics International s (CDI) icell Cardiac Progenitor Cells are a highly pure population of human cardiac progenitor cells derived from induced pluripotent stem (ips) cells using CDI s proprietary differentiation and purification protocols. icell Cardiac Progenitor Cells exhibit expected physiological characteristics and responses. These cells provide a reliable source of human cardiac progenitor cells suitable for use in targeted drug discovery, toxicity testing, and other life science research. A B Hoechst NKX2.5 CKIT 88% ± 2% Figure 1: icell Cardiac Progenitor Cells Represent a Highly Pure Population of Human Cardiac Progenitors These images show icell Cardiac Progenitor Cells at day 2 post-plating. icell Cardiac Progenitor Cells maintain KDR + /CKIT and NKX2.5 + profile as demonstrated by (A) flow cytometry and (B) immunocytochemistry: NKX2.5 (green) and nuclear staining Hoechst (blue). icell Cardiac Progenitor Cells Prototype User s Guide 1

6 Components Supplied by Cellular Dynamics Notes Item icell Cardiac Progenitor Cells Prototype 1 icell Cardiac Progenitor Cells Prototype User s Guide 1 Catalog Number CPC PT Certificate of Testing 2 Certificate of Origin If required for shipping purposes 1 Safety Data Sheet and User s Guide available online at 2 Available online at Required Equipment and Consumables Note: As required for the intended use, see the following icell Cardiac Progenitor Cells Prototype Application Protocols for assay-specific equipment and consumables before thawing cells: Modeling Cardiac Proliferation: bfgf Induction with High Content Analysis Modeling Cardiomyocyte Differentiation: Wnt- and Activin/TGFβ-inhibitor Induction with Flow Cytometry Analysis These Application Protocols are available online at Item Vendor Catalog Number Equipment 37 C Water Bath Biological Safety Cabinet with UV Lamp Cell Culture Incubator Hemocytometer or Automated Cell Counter* Liquid Nitrogen Storage Unit Pipettors Tabletop Centrifuge Consumables 15 ml and 50 ml Centrifuge Tubes 96-well Cell Culture Plates Cocktail B from Hepatocyte Maintenance Supplement Pack (Supplement Pack) Dulbecco s Phosphate Buffered Saline without Ca 2+ and Mg 2+ (D-PBS) Life Technologies CM4000 Fibronectin Roche Applied Sciences (1 mg) (5 mg) Gentamicin, 50 mg/ml Life Technologies Pipettes Sterile Tissue Culture Grade Distilled Water William s E Medium Life Technologies A * Ensure the automated cell counter is appropriately calibrated before use. 2 Cellular Dynamics International, Inc.

7 Notes Technical Support and Training CDI s Technical Support Scientists have the necessary laboratory and analytical experience to respond to your inquiries. In addition, in-lab training may be available upon request. Telephone Fax (608) (877) (US toll-free) / (608) x5 Monday - Friday, 8:30 am - 5:00 pm US Central Time support@cellulardynamics.com icell Cardiac Progenitor Cells Prototype User s Guide 3

8 Workflow Diagram Notes Upon receipt, immediately transfer to LN2 storage! Handling and Storage p. 5 Preparing Cell Culture Surfaces p. 6 Preparing the Medium p. 7 Culture Preparation Thawing p. 8 Plating for Proliferation p. 10 Plating for Differentiation p. 11 Applications Experimental Utilization (native biology, drug discovery, disease modeling, functionality, etc.) 4 Cellular Dynamics International, Inc.

9 Notes Chapter 2. Handling and Storage icell Cardiac Progenitor Cells are provided as cryopreserved single-cell suspensions in 1.5 ml cryovials. Upon receipt, directly transfer the cryobox containing icell Cardiac Progenitor Cells to the vapor phase of a liquid nitrogen storage dewar. CDI strongly recommends transferring the entire cryobox into the storage rack to avoid transferring individual vials. It is critical to maintain cryopreserved icell Cardiac Progenitor Cells at a stable temperature. Minimize exposure of cryopreserved icell Cardiac Progenitor Cells to ambient temperature when transferring vials to liquid nitrogen storage. icell Cardiac Progenitor Cells Prototype User s Guide 5

10 Chapter 3. Preparing Cell Culture Surfaces Notes icell Cardiac Progenitor Cells will function on cell culture vessels pre-coated with fibronectin. The following procedure details coating 96-well cell culture plates. Scale volumes appropriately for other vessel formats. 1. Dilute 1 mg/ml fibronectin solution in sterile D-PBS to a final concentration of 5 µg/ml immediately before use. Note: Reconstitute fibronectin in sterile water at 1 mg/ml according to the manufacturer s instructions. Aliquot and store at -20 C. 2. Add 100 µl/well of the 5 µg/ml fibronectin solution to the 96-well cell culture plate(s). 3. Incubate the cell culture plate(s) at 37 C for at least 1 hour before plating icell Cardiac Progenitor Cells. 6 Cellular Dynamics International, Inc.

11 Notes Chapter 4. Preparing the Medium The Maintenance Medium for icell Cardiac Progenitor Cells is comprised of William s E Medium, Cocktail B, and gentamicin. The Maintenance Medium is serum-free. 1. Prepare the Maintenance Medium by diluting Cocktail B in William s E Medium to 1X immediately before use. Note: Cocktail B is provided in the Supplement Pack and supplies the cardiac progenitor cells with a source of energy in the serum-free William s E Medium. Also provided in the Supplement Pack is dexamethasone, which is not used for preparing the Maintenance Medium. Note: Stored separately, William s E Medium and Cocktail B are stable at 4 C for 1 year according to the manufacturer. 2. Dilute gentamicin in Maintenance Medium at a final concentration of 25 μg/ml. 3. Invert to mix. Do not filter. 4. Store the Maintenance Medium at 4 C, protected from light, for up to 2 weeks. icell Cardiac Progenitor Cells Prototype User s Guide 7

12 Chapter 5. Thawing icell Cardiac Progenitor Cells Notes Maintain icell Cardiac Progenitor Cells in liquid nitrogen until immediately before thawing to ensure maximal performance of the cells. Complete the following steps of the thawing procedure in a time-efficient manner to facilitate optimal icell Cardiac Progenitor Cells viability and performance. See Chapters 6 and 7 for plating instructions for inducing proliferation or differentiation, respectively. Note: Thaw no more than 3 vials of icell Cardiac Progenitor Cells at one time. 1. Equilibrate the Maintenance Medium at room temperature for 2-4 hours before thawing icell Cardiac Progenitor Cells. 2. Remove the icell Cardiac Progenitor Cells cryovial from the liquid nitrogen storage tank. Note: If necessary, place cryovials on dry ice for up to 10 minutes before thawing. 3. Immerse the cryovial in a 37 C water bath for 4 minutes (avoid submerging the cap) holding the tube stationary (no swirling). Use of a floating microcentrifuge tube rack is recommended. 4. Immediately remove the cryovial from the water bath, spray with 70% ethanol, and place into the biological safety cabinet. 5. Gently transfer the icell Cardiac Progenitor Cells cryovial contents to a 50 ml centrifuge tube using a 1 ml pipettor. Note: Use of a 50 ml centrifuge tube facilitates suitable mixing to minimize osmotic shock and increase cardiac progenitor cell viability. Avoid repeated pipetting of the thawed icell Cardiac Progenitor Cells suspension. 6. Rinse the empty icell Cardiac Progenitor Cells cryovial with 1 ml of room temperature Maintenance Medium to recover residual cells from the cryovial. Transfer the 1 ml of Maintenance Medium rinse from the cryovial drop-wise (~1 drop every 4-5 seconds) to the 50 ml centrifuge tube containing the icell Cardiac Progenitor Cells suspension. Gently swirl the tube while adding the medium to mix the solution completely and minimize the osmotic shock on the thawed cells. Drop-wise addition of Maintenance Medium to the cell suspension is critical to minimize osmotic shock and ensure maximum viability and subsequent attachment of the cells to the plating substrate. 8 Cellular Dynamics International, Inc.

13 Notes 7. Add 3 ml of room temperature Maintenance Medium to the 50 ml centrifuge tube. Add the first 1 ml drop-wise over seconds. Then add the remaining 2 ml over the next ~30 seconds. Gently swirl the centrifuge tube while adding the medium. It is critical to add the 3 ml of Maintenance Medium slowly to ensure maximum viability and attachment of the cells once plated. 8. Gently mix the contents of the 50 ml centrifuge tube by inverting 2-3 times. Gentle mixing is critical to ensure maximum viability. Avoid vigorous shaking or vortexing of the cell suspension. Note: Thaw up to 3 vials of icell Cardiac Progenitor Cells at one time. Once thawed, combine the contents of the cryovials before adding the rinse and final volume of Maintenance Medium. Follow the timing outlined in steps 6 and 7. For example, if pooling 3 cryovials, add each 1 ml of rinse over 90 seconds (270 seconds total). icell Cardiac Progenitor Cells Prototype User s Guide 9

14 Chapter 6. Plating icell Cardiac Progenitor Cells for Cardiac Proliferation Notes icell Cardiac Progenitor Cells will proliferate when cultured in fibroblast growth factor (FGF)-containing Maintenance Medium for 2 days. The proliferation rate can be determined and quantified as number of NKX2.5 + cells. For assay instructions, see the icell Cardiac Progenitor Cells Prototype Application Protocol: Modeling Cardiac Proliferation: bfgf Induction with High Content Analysis available online at The following procedure describes how to plate icell Cardiac Progenitor Cells at 0.78 x 10 5 viable cells/cm 2 into a 96-well cell culture plate. Scale volumes appropriately for other cell culture vessel formats. 1. Invert the thawed icell Cardiac Progenitor Cells suspension 2-3 times to ensure an even cell distribution before performing the cell count. 2. Remove a sample of cells to perform a cell count using a hemocytometer (using trypan blue exclusion to identify viable cells) or an automated cell counter. 3. Dilute the cell suspension in room temperature Maintenance Medium to 2.8 x 10 5 viable cells/ml. 4. Aspirate the fibronectin solution from a pre-coated 96-well cell culture plate. 5. Invert the cell suspension 6 times. Immediately dispense 90 µl/well of cell suspension (~25,000 viable cells/well). 6. Immediately proceed to the instructions in the icell Cardiac Progenitor Cells Prototype Application Protocol: Modeling Cardiac Proliferation: bfgf Induction with High Content Analysis for cell culturing with bfgf and labeling for analysis. 10 Cellular Dynamics International, Inc.

15 Notes Chapter 7. Plating icell Cardiac Progenitor Cells for Cardiomyocyte Differentiation icell Cardiac Progenitor Cells will differentiate into cardiomyocytes within 6-8 days when cultured in XAV939/SB containing Maintenance Medium for 2 days, then in XAV939/SB free Maintenance Medium for 4-6 days. The differentiation can be determined and quantified as number of cardiac troponin T (ctnt) + cells. For assay instructions, see the icell Cardiac Progenitor Cells Prototype Application Protocol: Modeling Cardiomyocyte Differentiation: Wnt- and Activin/TGFβ-inhibitor Induction with Flow Cytometry Analysis available online at The following procedure describes how to plate icell Cardiac Progenitor Cells at 1.56 x 10 5 viable cells/cm 2 into a 96-well cell culture plate. Scale volumes appropriately for other cell culture vessel formats. 1. Invert the thawed icell Cardiac Progenitor Cells suspension 2-3 times to ensure an even cell distribution before performing the cell count. 2. Remove a sample of cells to perform a cell count using a hemocytometer (using trypan blue exclusion to identify viable cells) or an automated cell counter. 3. Dilute the cell suspension in room temperature Maintenance Medium to 5.6 x 10 5 viable cells/ml. 4. Aspirate the fibronectin solution from a pre-coated 96-well cell culture plate. 5. Invert the cell suspension 6 times. Immediately dispense 90 µl/well of cell suspension (~50,000 viable cells/well). 6. Immediately proceed to the instructions in the icell Cardiac Progenitor Cells Prototype Application Protocol: Modeling Cardiomyocyte Differentiation: Wntand Activin/TGFβ-inhibitor Induction with Flow Cytometry Analysis for cell culturing with XAV939 and SB431542, collecting, staining, and labeling for analysis. icell Cardiac Progenitor Cells Prototype User s Guide 11

16 Appendices Notes Appendix A. Intellectual Property Rights, Use Restrictions, and Limited License A. OWNERSHIP. The Products are covered by pending patents and patents: cellulardynamics.com/about-us/patents/. Customer has a limited license to use the Products for internal research purposes for the sole benefit of the Customer, subject to the use restrictions included in subsection B of this Appendix A. Customer acknowledges and agrees that the receipt or purchase of the Products by Customer shall not be construed as a transfer of any title or the grant of any rights in or to the intellectual property embodied in the Products owned or licensed by Cellular Dynamics. In particular, no right or license to make, have made, offer to sell, or sell the Products, to modify or reproduce the Product or any part thereof, or to use the Products in combination with any other product(s), except product(s) provided or expressly licensed to Customer by Cellular Dynamics for such use, is implied or conveyed by the sale or transfer of Products to Customer. B. USE RESTRICTIONS. The Products are licensed for internal research purposes only, and may not be used for any other purpose. The Products must be used in accordance with this User s Guide to which Customer, by ordering and accepting the Products, agrees. Customer shall not make, have made, offer to sell, or sell the Products. Unless expressly permitted in a contract executed between Customer and Cellular Dynamics, Customer shall not use the Products (or any modifications Customer makes to the Products or any cells derived, developed or expanded from the Products) in (i) the manufacture of any products, or (ii) any services for a third party. Customer may not transfer the Products (or any modifications Customer makes to the Products or any cells derived, developed or expanded from the Products) to any third party without Cellular Dynamics prior written consent. Customer shall not reverse engineer the Products. Customer shall not use the Products, components or modifications thereof, or any cells derived, developed or expanded therefrom, in humans, in clinical trials, for diagnostic purposes involving human subjects, or for any investigational or other therapeutic use. Customer shall not use the Products directly or indirectly to derive or make any human gamete or gamete precursor cell. Customer shall use the Products in accordance with all applicable laws and regulations and any applicable institutional review board approved protocol and/or privacy office approval. Customer is not entitled to receive any data or information from Cellular Dynamics that directly identifies the donor of the biological materials from which the Products indirectly are derived or were made. Customer shall not attempt in any way to determine the identity of the donor of the biological materials from which the Products indirectly are derived or were made. C. DATA. Customer agrees that if described on Customer s product quotation from Cellular Dynamics it will provide data and information as described therein to Cellular Dynamics regarding Customer s use of the Products. Appendix B. Product Provided AS IS A. The Products are sold or provided AS IS. B. TO THE FULLEST EXTENT PERMITTED BY APPLICABLE LAW, CELLULAR DYNAMICS DISCLAIMS, ALL REPRESENTATIONS, AND WARRANTIES, EXPRESS OR IMPLIED (INCLUDING BUT NOT LIMITED TO, ANY IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE OR NON- INFRINGEMENT), AND ALL LIABILITY, WITH RESPECT TO THE PRODUCTS; AND BY ORDERING AND ACCEPTING THE PRODUCTS CUSTOMER WAIVES ALL RIGHTS AND REMEDIES OTHERWISE AVAILABLE WITH RESPECT TO THE PRODUCTS. 12 Cellular Dynamics International, Inc.

17 Notes C. Customer will be solely responsible for (i) Customer s use of the Products for a purpose or in a manner other than that for which they were designed or that is permitted or in breach of the Use Restrictions above; (ii) Customer s failure to follow this User s Guide for the use, storage, and handling of the Products however such failure is caused; (iii) Customer s failure to comply with any of the provisions of Appendix A above; and (iv) any abuse, other misuse or neglect of the Products by Customer or any damage or loss of the Products by events or occurrences beyond a person s (e.g., Cellular Dynamics ) control including without limitation, accident, fire, vandalism and natural disasters (acts of God). D. Customer acknowledges and agrees that Cellular Dynamics may fill Customer s order with any number of units of Products. Such units may be more units than Customer ordered. Customer will not be charged extra for any adjustments made by Cellular Dynamics. Because the number of cells in a unit may vary from lot to lot, Cellular Dynamics reserves the right to fill the order with that number of units which is sufficient to fill Customer s order. Appendix C. Limited Liability TO THE FULLEST EXTENT PERMITTED UNDER APPLICABLE LAW, CELLULAR DYNAMICS SHALL NOT HAVE ANY LIABILITY FOR INCIDENTAL, COMPENSATORY, PUNITIVE, CONSEQUENTIAL, INDIRECT, SPECIAL OR OTHER SIMILAR DAMAGES, HOWEVER CAUSED AND REGARDLESS OF FORM OF ACTION WHETHER IN CONTRACT, TORT (INCLUDING NEGLIGENCE), STRICT PRODUCT LIABILITY OR OTHERWISE, EVEN IF CELLULAR DYNAMICS HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. NOTWITHSTANDING ANY OTHER TERM OR IMPLICATION TO THE CONTRARY, UNDER NO CIRCUMSTANCES SHALL CELLULAR DYNAMICS LIABILITY TO CUSTOMER EXCEED THE AMOUNT PAID BY CUSTOMER FOR THE PRODUCTS TO CELLULAR DYNAMICS Cellular Dynamics International, Inc. All rights reserved. icell Cardiac Progenitor Cells Prototype User s Guide 13